Supplemental Methods
Fluorescence-activated cell sorting and PIK3CA sequencing of AR
expressing TNBC cell lines. AR positive cell lines (1 x 106) were harvested
and fixed in 4% paraformaldehyde followed by permeabliization in cold 90%
methanol.
Cells were incubated with anti-AR antibody (clone# D6F11, Cell
Signaling) (1:100) at room temperature (RT) for 1hr. After a PBS wash cells
were incubated with goat anti-rabbit Alexaflour 488 antibody (invitrogen)
(1:10,000) at RT for 30 min. CAL-148 cells (5E5) were sorted into ARlow and
ARhigh populations and DNA extracted (DNA easy, Qiagen). PCR was performed
on PIK3CA amplicons similar to above (see PIK3CA mutation evaluation)
followed by Sanger sequencing.
Quantification of apoptosis.
Caspase 3/7 activity.
Cell lines were seeded in triplicate in 96-well plates. Media was removed and
replaced with media containing vehicle (control) or indicated drugs. After 48 h,
apoptosis was determined by measuring luciferase from activated caspase 3/7
after addition of Caspase-Glo reagent (Promega). Relative levels of caspase
activity were normalized to viable cell number determined by metabolic reduction
of alamarBlue.
Cell cycle quantification of DNA fragmentation.
Cells were seeded in 60mm dishes (1 x 106 cells/dish) in appropriate
supplemented medium.
Twenty-four hours later, cells were incubated for an
additional 48h in the presence of CDX (25 M), GDC-0941 (1 M), or GDC-0980
(300 nM). All drugs were added individually as well as in combinations that
included CDX with either GDC-0941 or GDC-0980. ADR (3 M) was used as a
positive control. Cell medium was collected while adherent cells were harvested
and resuspended in 1 ml cold PBS. To fix cells, 0.7 ml cold ethanol (70%) was
added dropwise to each tube while vortexing gently.
After samples were
incubated overnight at 4 °C , they were washed once with 1 ml PBS, and
resuspended in 400 l PI/Triton X-100 staining solution (0.1% (v/v) Triton X-100
(Sigma) in PBS containing 200 ng/ml DNAse-free RNAse A (Sigma) and 40
g/ml propidium iodide (PI). Cells were incubated at 37°C for 15 min and then
transferred to ice and protected from light until fluorescence was read by flow
cytometer (3-laser Becton Dickinson LSRII).
Immunofluorescence.
Cells
were
fixed
(4%
paraformaldehyde)
and
permeablized (90% cold methanol) prior to 1 h blocking in 1% BSA. Cells were
incubated with rabbit anti-AR (1:500, Cell Signaling, D6F11) overnight and then
washed with PBS and incubated with anti-rabbit Alexa Flour 488 (1:10:000,
invitrogen) for 1 h. Cells were then washed and blocked with rabbit Fab (2)
fragments (1:30) for 1 h. Following a wash, cells were incubated in rabbit anti-pAKT (1:50) overnight. Cells were washed and incubated with anti-rabbit Alexa
Flour 594 (1:10,000) for 1 h followed by a wash. Cells were cytospun onto slides
and mounted in DAPI containing mounting media.